vegf a165  (R&D Systems)

 
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    Name:
    Recombinant Human VEGF 165b Protein CF
    Description:
    The Recombinant Human VEGF 165b Protein from R D Systems is derived from Sf 21 stably transfected The Recombinant Human VEGF 165b Protein has been validated for the following applications Bioactivity
    Catalog Number:
    3045-VE-025/CF
    Price:
    329
    Category:
    Proteins and Enzymes
    Source:
    Sf 21 (stably transfected)-derived Recombinant Human VEGF 165b Protein
    Applications:
    Bioactivity
    Purity:
    >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
    Conjugate:
    Unconjugated
    Size:
    25 ug
    Buy from Supplier


    Structured Review

    R&D Systems vegf a165
    The Dynamic Curves of <t>VEGF</t> inhibitors with VEGF isoforms and PlGF detected by BIACORE. VEGF isoforms and PlGF were set as ligands to be immobilized onto the CM5 chips. Different VEGF inhibitors were set as analytes and two-fold diluted into six or eight concentrations. (A) conbercept with <t>VEGF-A165</t> (B) conbercept with VEGF-A121. (C) Avastin with VEGF-A165. (D) Avastin with VEGF-A121. (E) conbercept with VEGF-B167. (F) conbercept with PlGF.
    The Recombinant Human VEGF 165b Protein from R D Systems is derived from Sf 21 stably transfected The Recombinant Human VEGF 165b Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/vegf a165/product/R&D Systems
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vegf a165 - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "Novel VEGF Decoy Receptor Fusion Protein Conbercept Targeting Multiple VEGF Isoforms Provide Remarkable Anti-Angiogenesis Effect In Vivo"

    Article Title: Novel VEGF Decoy Receptor Fusion Protein Conbercept Targeting Multiple VEGF Isoforms Provide Remarkable Anti-Angiogenesis Effect In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070544

    The Dynamic Curves of VEGF inhibitors with VEGF isoforms and PlGF detected by BIACORE. VEGF isoforms and PlGF were set as ligands to be immobilized onto the CM5 chips. Different VEGF inhibitors were set as analytes and two-fold diluted into six or eight concentrations. (A) conbercept with VEGF-A165 (B) conbercept with VEGF-A121. (C) Avastin with VEGF-A165. (D) Avastin with VEGF-A121. (E) conbercept with VEGF-B167. (F) conbercept with PlGF.
    Figure Legend Snippet: The Dynamic Curves of VEGF inhibitors with VEGF isoforms and PlGF detected by BIACORE. VEGF isoforms and PlGF were set as ligands to be immobilized onto the CM5 chips. Different VEGF inhibitors were set as analytes and two-fold diluted into six or eight concentrations. (A) conbercept with VEGF-A165 (B) conbercept with VEGF-A121. (C) Avastin with VEGF-A165. (D) Avastin with VEGF-A121. (E) conbercept with VEGF-B167. (F) conbercept with PlGF.

    Techniques Used:

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Biomarkers for antitumor activity of bevacizumab in gastric cancer models
    Article Snippet: Human VEGF, placental growth factor (Pl GF), interleukin-8 (IL-8) and basic fibroblast growth factor (bFGF) were quantified using Quantikine® ELISA kits (R & D Systems, Minneapolis, MN, USA). .. Human VEGF165b was quantified using the DuoSet® ELISA Development System (R & D Systems). ..

    Article Title: VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy
    Article Snippet: Thus the efficiency of amplification of the two templates is not different. shows the amplification curves for the exon 8a primers using the VEGF165 sequence as a template (VEGF165 b template did not result in amplification until 18 cycles later than equivalent VEGF165 concentration). .. The commercially available pan-VEGF ELISA has a lower affinity for VEGF165 b than for VEGF165 by 42%. .. Therefore the measured VEGF levels (VEGFmeasured ) in the commercially available ELISA are the sum of VEGF165 and 42% of VEGF165b.

    Article Title: VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy
    Article Snippet: Regeneration between each interaction was performed by injection of 4 mol l−1 MgCl2 at 20 μ l min−1 for 40 s, followed by a 2-min period of stabilisation before the next injection. shows the binding curves of VEGF165 to the RnD detection antibody and shows binding of VEGF165 b to the same antibody. .. The RnD detection antibody had a higher association coefficient for VEGF165 than VEGF165 b and a lower dissociation coefficient for VEGF165 than VEGF165 b , resulting in an affinity of 602 p M for VEGF165 , but 3.98 n M for VEGF165 b, an ∼6.6-fold difference in affinity, indicating that the underestimation of the commercial ELISA for VEGF165b was due to a difference in affinity for the antigen. .. Thus the efficiency of amplification of the two templates is not different. shows the amplification curves for the exon 8a primers using the VEGF165 sequence as a template (VEGF165 b template did not result in amplification until 18 cycles later than equivalent VEGF165 concentration).

    Recombinant:

    Article Title: Neutralization of vascular endothelial growth factor antiangiogenic isoforms or administration of proangiogenic isoforms stimulates vascular development in the rat testis
    Article Snippet: The VEGFA primary antibody was diluted 1:100, the VEGFAxxxB antibody was diluted 1:1,000, and the secondary antibodies were diluted 1:6,000 in blocking buffer. .. Recombinant mouse VEGFA_164 and VEGFA_165B (R & D Systems, Minneapolis, MN) were utilized as loading controls for VEGFA antibody and VEGFAxxxB antibody blots, respectively. .. Images of blots were acquired with the Odyssey infrared imaging system and the Odyssey software program (LI-COR Biosciences) was utilized for quantitative analysis.

    Multiple Displacement Amplification:

    Article Title: Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer
    Article Snippet: .. U87 or MDA-MB-231 cells were cultured, in bevacizumab (Genentech), HGF (294-HGN-005/CF, R & D Systems), hypoxia, conditioned HUVEC media, or serum-free media containing VEGF165 (584501, Biolegend), VEGF165b (3045VE025CF, R & D Systems), or VEGF189 (8147VE025CF, R & D Systems) and complex formation was assessed via IP. c-Met activation through β1 signaling was assessed by plating U87 cells on increasing fibronectin amounts (sc-29011, Santa Cruz Biotechnology) and blotting for phosphorylated c-Met. .. Transduced MDA-MB-231 or U87 cells were plated in six-well plates at 90% confluency.

    Cell Culture:

    Article Title: Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer
    Article Snippet: .. U87 or MDA-MB-231 cells were cultured, in bevacizumab (Genentech), HGF (294-HGN-005/CF, R & D Systems), hypoxia, conditioned HUVEC media, or serum-free media containing VEGF165 (584501, Biolegend), VEGF165b (3045VE025CF, R & D Systems), or VEGF189 (8147VE025CF, R & D Systems) and complex formation was assessed via IP. c-Met activation through β1 signaling was assessed by plating U87 cells on increasing fibronectin amounts (sc-29011, Santa Cruz Biotechnology) and blotting for phosphorylated c-Met. .. Transduced MDA-MB-231 or U87 cells were plated in six-well plates at 90% confluency.

    Activation Assay:

    Article Title: Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer
    Article Snippet: .. U87 or MDA-MB-231 cells were cultured, in bevacizumab (Genentech), HGF (294-HGN-005/CF, R & D Systems), hypoxia, conditioned HUVEC media, or serum-free media containing VEGF165 (584501, Biolegend), VEGF165b (3045VE025CF, R & D Systems), or VEGF189 (8147VE025CF, R & D Systems) and complex formation was assessed via IP. c-Met activation through β1 signaling was assessed by plating U87 cells on increasing fibronectin amounts (sc-29011, Santa Cruz Biotechnology) and blotting for phosphorylated c-Met. .. Transduced MDA-MB-231 or U87 cells were plated in six-well plates at 90% confluency.

    Transfection:

    Article Title: VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy
    Article Snippet: Vascular endothelial growth factor concentrations of the LS174t human colonic carcinoma cell line transfected to overexpress VEGF165 , VEGF165 b, VEGF165 and VEGF165 b (VEGF165/165b), or with the empty expression vector (pcDNA3) are given in . .. Proliferation of LS174t cells after transfection with VEGF165 , VEGF165 b, both or control vector was measured by flow cytometry. .. Reverse transcription-polymerase chain reaction gave two bands, one at ∼135 bp, consistent with VEGF165 b or VEGF189 b, and one at ∼200 bp, consistent with VEGF165 and VEGF189 .

    Plasmid Preparation:

    Article Title: VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy
    Article Snippet: Vascular endothelial growth factor concentrations of the LS174t human colonic carcinoma cell line transfected to overexpress VEGF165 , VEGF165 b, VEGF165 and VEGF165 b (VEGF165/165b), or with the empty expression vector (pcDNA3) are given in . .. Proliferation of LS174t cells after transfection with VEGF165 , VEGF165 b, both or control vector was measured by flow cytometry. .. Reverse transcription-polymerase chain reaction gave two bands, one at ∼135 bp, consistent with VEGF165 b or VEGF189 b, and one at ∼200 bp, consistent with VEGF165 and VEGF189 .

    Flow Cytometry:

    Article Title: VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy
    Article Snippet: Vascular endothelial growth factor concentrations of the LS174t human colonic carcinoma cell line transfected to overexpress VEGF165 , VEGF165 b, VEGF165 and VEGF165 b (VEGF165/165b), or with the empty expression vector (pcDNA3) are given in . .. Proliferation of LS174t cells after transfection with VEGF165 , VEGF165 b, both or control vector was measured by flow cytometry. .. Reverse transcription-polymerase chain reaction gave two bands, one at ∼135 bp, consistent with VEGF165 b or VEGF189 b, and one at ∼200 bp, consistent with VEGF165 and VEGF189 .

    Angiogenesis Assay:

    Article Title: VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy
    Article Snippet: To confirm that the cytotoxicity effects of anti-VEGF antibodies were due to inhibition of VEGFxxx b, the effects of supplementing the media with rhVEGF165 b were measured ( ). .. Furthermore, the ability of VEGF165 b to inhibit VEGF165 -mediated angiogenesis in a rat mesenteric and rabbit corneal assay ( ) and mouse retinal angiogenesis assay ( ) suggests that it may serve as a novel AAT agent. .. Consistent with this, we show here that the overexpression of VEGF165 b in human CRC can inhibit tumour growth in vivo , further supporting the potential role of relative VEGFxxx b downregulation as part of the angiogenic switch in tumour progression.

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  • 94
    R&D Systems recombinant vegf
    HUVEC response to soluble morphogens. HUVECs starved overnight in 2% <t>FBS</t> were seeded on arrays presenting varied densities of GRGDSP in the presence of 2% FBS alone or with the addition of 10 ng/mL VR-BP (4.8 nM) or 10 ng/mL <t>VEGF</t> (0.26 nM). (Error bars indicate standard error of the mean and asterisk indicate significance compared to FBS alone, p
    Recombinant Vegf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant vegf/product/R&D Systems
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant vegf - by Bioz Stars, 2021-07
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    99
    R&D Systems biotinylated vegf a165
    Binding of AGuIX@PS, AGuIX@PS@scramble and AGuIX@PS@KDKPPR NPs to recombinant NRP-1 protein. Notes: ( A ) Binding of <t>biotinylated</t> <t>VEGF</t> 165 (5 ng/mL; 110 pM) to NRP-1 in the presence of 2 µg/mL heparin was evaluated when increasing concentrations of peptide (6.5–200 µM) were added (data points show the mean ± SD, n=3). ( B ) AGuIX@PS@KDKPPR NPs lead to a relevant affinity toward NRP-1 protein compared to maleimido-PS@KDKPPR or AGuIX@PS@scramble. Abbreviations: PS, photosensitizer; NP, nanoparticle; EC 50 , concentration of competitor that displaced 50% of biotinylated VEGF-A 165 binding.
    Biotinylated Vegf A165, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated vegf a165/product/R&D Systems
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    93
    R&D Systems biotinylated bt vegf a165
    Binding of <t>VEGF-A</t> 165 -HBD to NRP as detected by a cell based assay and size exclusion chromatography. (a) Binding of VEGF-A 165 -HBD to NRP1 was determined in DU145 cells expressing an adenoviral construct encoding NRP1 [17] . These cells do not express other VEGF receptors [21] . Cells were incubated with <t>biotinylated</t> VEGF-A 165 in the presence of the indicated concentrations of either unlabelled VEGF-A 165 (positive control), or VEGF-A 165 -HBD containing a His 6 tag or VEGF-A 165 -HBD without the tag. Values presented are the means (±SEM) obtained from two independent experiments. Other experimental details are described in Materials and Methods. (b) Binding of VEGF-A 165 -HBD to b1 domains of NRP1 and NRP2 was assessed by size exclusion chromatography. The protein mixtures of a tenfold molar excess of VEGF-A 165 -HBD with the purified b1 domain from either NRP1 or NRP2 were incubated at room temperature for 30 minutes. The FPLC profile for VEGF-A 165 -HBD and NRP1 b1 mixture is shown. SDS-PAGE analysis was used to evaluate samples of NRP1 b1, VEGF-A 165 -HBD, and the protein mixture before being loaded onto the size exclusion column (lanes 1–3, respectively) as well as samples of fractions which eluted from the single peak (lanes 4–6), showing that VEGF-A 165 -HBD and NRP1 b1 co-eluted from the preparative Superdex 75 column. A similar result was seen for VEGF-A 165 -HBD and NRP2 b1. (c) Formation of the molecular complexes was also investigated by analytical size exclusion chromatography. NRP1 b1 and b1b2 domains as well VEGF-A 165 -HBD were initially loaded separately onto the analytical Superdex 75 column and their corresponding FPLC traces are shown in solid and dashed lines, respectively. To detect binding, NRP1 b1 or b1b2 domains were mixed with VEGF-A 165 -HBD in solution at a molar ratio of 1∶10 and 1∶28, respectively and incubated for an hour at room temperature. The mixtures were then applied to the column. The main peaks in the elution profiles revealed a shift to the left of the peak positions corresponding to the unbound NRP1 b1 or b1b2 domains (indicated by vertical black lines), suggesting complex formation.
    Biotinylated Bt Vegf A165, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated bt vegf a165/product/R&D Systems
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotinylated bt vegf a165 - by Bioz Stars, 2021-07
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    98
    R&D Systems recombinant human vegf a165
    Delivering GFs and laminin HBD peptide enhances skin wound healing. Full-thickness back-skin wounds in 10– 11-week-old C57BLKS/J-m/Lepr db (db/db) mice were treated with combined <t>VEGF-A165</t> (100 ng/wound) and PDGF-BB (50 ng/wound). Four groups were tested: fibrin only, fibrin functionalized with α 2 PI 1–8 -LAMA3 3043–3067 peptide, fibrin containing admixed GFs, and fibrin functionalized with α 2 PI 1–8 -LAMA3 3043–3067 peptide containing GFs. After 4, 7, and 10 days, a , b wound closure and c granulation tissue area were evaluated by histology (means ± SEM, day 4: n = 6, day 7: fibrin only, and α 2 PI 1–8 -LAMA3 3043–3067 peptide + GFs, n = 10; other treatment groups, n = 11, day 10: α 2 PI 1–8 -LAMA3 3043–3067 peptide, n = 8, α 2 PI 1–8 -LAMA3 3043–3067 peptide + GFs, n = 9, and other treatment groups, n = 7). b The proportions of the mice were categorized by the degree of healing after day 7 of wound treatment. d Wound histology (hematoxylin and eosin staining) at day 7. Red arrows indicate tips of the epithelium tongue. The granulation tissue (pink–violet) is characterized by a large number of granulocytes with nuclei that stain in dark-violet or black. Muscle under the wounds is stained in red. Fat tissue appears as transparent bubbles. Scale bar = 800 µm. e – g A total of 5 days after the wound treatment, e proliferation of CD31 + CD45 – endothelial cells is assessed by Ki67 + marker, and f the frequency of Ly6G + CD11b + neutrophils within CD45 + cells and g the frequency of Ly6C + CD11b + monocytes within CD45 + cells were determined using flow cytometry (means ± SEM). * P
    Recombinant Human Vegf A165, supplied by R&D Systems, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human vegf a165/product/R&D Systems
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human vegf a165 - by Bioz Stars, 2021-07
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    Image Search Results


    HUVEC response to soluble morphogens. HUVECs starved overnight in 2% FBS were seeded on arrays presenting varied densities of GRGDSP in the presence of 2% FBS alone or with the addition of 10 ng/mL VR-BP (4.8 nM) or 10 ng/mL VEGF (0.26 nM). (Error bars indicate standard error of the mean and asterisk indicate significance compared to FBS alone, p

    Journal: Integrative biology : quantitative biosciences from nano to macro

    Article Title: Differential effects of a soluble or immobilized VEGFR-binding peptide

    doi: 10.1039/c2ib20055d

    Figure Lengend Snippet: HUVEC response to soluble morphogens. HUVECs starved overnight in 2% FBS were seeded on arrays presenting varied densities of GRGDSP in the presence of 2% FBS alone or with the addition of 10 ng/mL VR-BP (4.8 nM) or 10 ng/mL VEGF (0.26 nM). (Error bars indicate standard error of the mean and asterisk indicate significance compared to FBS alone, p

    Article Snippet: After imaging, media was replaced with m199 with 2% FBS containing varied concentrations soluble factors such as recombinant VEGF (VEGF-A165 , R & D systems, Minneapolis, MN) or VR-BP.

    Techniques:

    Binding of AGuIX@PS, AGuIX@PS@scramble and AGuIX@PS@KDKPPR NPs to recombinant NRP-1 protein. Notes: ( A ) Binding of biotinylated VEGF 165 (5 ng/mL; 110 pM) to NRP-1 in the presence of 2 µg/mL heparin was evaluated when increasing concentrations of peptide (6.5–200 µM) were added (data points show the mean ± SD, n=3). ( B ) AGuIX@PS@KDKPPR NPs lead to a relevant affinity toward NRP-1 protein compared to maleimido-PS@KDKPPR or AGuIX@PS@scramble. Abbreviations: PS, photosensitizer; NP, nanoparticle; EC 50 , concentration of competitor that displaced 50% of biotinylated VEGF-A 165 binding.

    Journal: International Journal of Nanomedicine

    Article Title: Ultrasmall AGuIX theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of glioblastoma

    doi: 10.2147/IJN.S141559

    Figure Lengend Snippet: Binding of AGuIX@PS, AGuIX@PS@scramble and AGuIX@PS@KDKPPR NPs to recombinant NRP-1 protein. Notes: ( A ) Binding of biotinylated VEGF 165 (5 ng/mL; 110 pM) to NRP-1 in the presence of 2 µg/mL heparin was evaluated when increasing concentrations of peptide (6.5–200 µM) were added (data points show the mean ± SD, n=3). ( B ) AGuIX@PS@KDKPPR NPs lead to a relevant affinity toward NRP-1 protein compared to maleimido-PS@KDKPPR or AGuIX@PS@scramble. Abbreviations: PS, photosensitizer; NP, nanoparticle; EC 50 , concentration of competitor that displaced 50% of biotinylated VEGF-A 165 binding.

    Article Snippet: In all, 5 ng/mL of biotinylated VEGF-A165 (R & D Systems, Inc.) in blocking buffer containing 2 µg/mL of heparin was used to assess the binding of compounds to NRP-1.

    Techniques: Binding Assay, Recombinant, Concentration Assay

    PS characterizations. Notes: ( A ) Absorption coefficient, fluorescence and singlet oxygen quantum yields of PS, PS@KDKPPR, Maleimido-PS@KDKPPR, Maleimido-PS@scramble. ( B ) Binding of the DKPPR peptide, the KDKPPR peptide, the PS@KDKPPR and the maleimido-PS@KDKPPR to recombinant NRP-1 protein. Binding of biotinylated VEGF (5 ng/mL; 110 pM) to NRP-1 in the presence of 2 µg/mL heparin was evaluated when increasing concentrations of compounds (6.5–200 µM) were added (data points show the mean ± SD, n=3). The values found for EC 50 are 46 µM for DKPPR, 2 µM for KDKPPR, 1 µM for PS@KDKPPR and 81 µM for maleimido-PS@KDKPPR. Abbreviations: PS, photosensitizer; DMSO, dimethyl sulfoxide; EC 50 , concentration of competitor that displaced 50% of biotinylated VEGF-A 165 binding.

    Journal: International Journal of Nanomedicine

    Article Title: Ultrasmall AGuIX theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of glioblastoma

    doi: 10.2147/IJN.S141559

    Figure Lengend Snippet: PS characterizations. Notes: ( A ) Absorption coefficient, fluorescence and singlet oxygen quantum yields of PS, PS@KDKPPR, Maleimido-PS@KDKPPR, Maleimido-PS@scramble. ( B ) Binding of the DKPPR peptide, the KDKPPR peptide, the PS@KDKPPR and the maleimido-PS@KDKPPR to recombinant NRP-1 protein. Binding of biotinylated VEGF (5 ng/mL; 110 pM) to NRP-1 in the presence of 2 µg/mL heparin was evaluated when increasing concentrations of compounds (6.5–200 µM) were added (data points show the mean ± SD, n=3). The values found for EC 50 are 46 µM for DKPPR, 2 µM for KDKPPR, 1 µM for PS@KDKPPR and 81 µM for maleimido-PS@KDKPPR. Abbreviations: PS, photosensitizer; DMSO, dimethyl sulfoxide; EC 50 , concentration of competitor that displaced 50% of biotinylated VEGF-A 165 binding.

    Article Snippet: In all, 5 ng/mL of biotinylated VEGF-A165 (R & D Systems, Inc.) in blocking buffer containing 2 µg/mL of heparin was used to assess the binding of compounds to NRP-1.

    Techniques: Fluorescence, Binding Assay, Recombinant, Protein Binding, Concentration Assay

    Binding of VEGF-A 165 -HBD to NRP as detected by a cell based assay and size exclusion chromatography. (a) Binding of VEGF-A 165 -HBD to NRP1 was determined in DU145 cells expressing an adenoviral construct encoding NRP1 [17] . These cells do not express other VEGF receptors [21] . Cells were incubated with biotinylated VEGF-A 165 in the presence of the indicated concentrations of either unlabelled VEGF-A 165 (positive control), or VEGF-A 165 -HBD containing a His 6 tag or VEGF-A 165 -HBD without the tag. Values presented are the means (±SEM) obtained from two independent experiments. Other experimental details are described in Materials and Methods. (b) Binding of VEGF-A 165 -HBD to b1 domains of NRP1 and NRP2 was assessed by size exclusion chromatography. The protein mixtures of a tenfold molar excess of VEGF-A 165 -HBD with the purified b1 domain from either NRP1 or NRP2 were incubated at room temperature for 30 minutes. The FPLC profile for VEGF-A 165 -HBD and NRP1 b1 mixture is shown. SDS-PAGE analysis was used to evaluate samples of NRP1 b1, VEGF-A 165 -HBD, and the protein mixture before being loaded onto the size exclusion column (lanes 1–3, respectively) as well as samples of fractions which eluted from the single peak (lanes 4–6), showing that VEGF-A 165 -HBD and NRP1 b1 co-eluted from the preparative Superdex 75 column. A similar result was seen for VEGF-A 165 -HBD and NRP2 b1. (c) Formation of the molecular complexes was also investigated by analytical size exclusion chromatography. NRP1 b1 and b1b2 domains as well VEGF-A 165 -HBD were initially loaded separately onto the analytical Superdex 75 column and their corresponding FPLC traces are shown in solid and dashed lines, respectively. To detect binding, NRP1 b1 or b1b2 domains were mixed with VEGF-A 165 -HBD in solution at a molar ratio of 1∶10 and 1∶28, respectively and incubated for an hour at room temperature. The mixtures were then applied to the column. The main peaks in the elution profiles revealed a shift to the left of the peak positions corresponding to the unbound NRP1 b1 or b1b2 domains (indicated by vertical black lines), suggesting complex formation.

    Journal: PLoS ONE

    Article Title: Production of Soluble Human Vascular Endothelial Growth Factor VEGF-A165-Heparin Binding Domain in Escherichia coli

    doi: 10.1371/journal.pone.0055690

    Figure Lengend Snippet: Binding of VEGF-A 165 -HBD to NRP as detected by a cell based assay and size exclusion chromatography. (a) Binding of VEGF-A 165 -HBD to NRP1 was determined in DU145 cells expressing an adenoviral construct encoding NRP1 [17] . These cells do not express other VEGF receptors [21] . Cells were incubated with biotinylated VEGF-A 165 in the presence of the indicated concentrations of either unlabelled VEGF-A 165 (positive control), or VEGF-A 165 -HBD containing a His 6 tag or VEGF-A 165 -HBD without the tag. Values presented are the means (±SEM) obtained from two independent experiments. Other experimental details are described in Materials and Methods. (b) Binding of VEGF-A 165 -HBD to b1 domains of NRP1 and NRP2 was assessed by size exclusion chromatography. The protein mixtures of a tenfold molar excess of VEGF-A 165 -HBD with the purified b1 domain from either NRP1 or NRP2 were incubated at room temperature for 30 minutes. The FPLC profile for VEGF-A 165 -HBD and NRP1 b1 mixture is shown. SDS-PAGE analysis was used to evaluate samples of NRP1 b1, VEGF-A 165 -HBD, and the protein mixture before being loaded onto the size exclusion column (lanes 1–3, respectively) as well as samples of fractions which eluted from the single peak (lanes 4–6), showing that VEGF-A 165 -HBD and NRP1 b1 co-eluted from the preparative Superdex 75 column. A similar result was seen for VEGF-A 165 -HBD and NRP2 b1. (c) Formation of the molecular complexes was also investigated by analytical size exclusion chromatography. NRP1 b1 and b1b2 domains as well VEGF-A 165 -HBD were initially loaded separately onto the analytical Superdex 75 column and their corresponding FPLC traces are shown in solid and dashed lines, respectively. To detect binding, NRP1 b1 or b1b2 domains were mixed with VEGF-A 165 -HBD in solution at a molar ratio of 1∶10 and 1∶28, respectively and incubated for an hour at room temperature. The mixtures were then applied to the column. The main peaks in the elution profiles revealed a shift to the left of the peak positions corresponding to the unbound NRP1 b1 or b1b2 domains (indicated by vertical black lines), suggesting complex formation.

    Article Snippet: The binding assay was performed 48 hours after adenoviral infection by the addition of various concentrations of either VEGF-A165 (R & D Systems) (positive control) or VEGF-A165 -HBD diluted in binding medium with or without 0.1% bovine serum albumin (BSA), followed by addition of 1 nM biotinylated (bt) VEGF-A165 (R & D Systems).

    Techniques: Binding Assay, Cell Based Assay, Size-exclusion Chromatography, Expressing, Construct, Incubation, Positive Control, Purification, Fast Protein Liquid Chromatography, SDS Page

    Delivering GFs and laminin HBD peptide enhances skin wound healing. Full-thickness back-skin wounds in 10– 11-week-old C57BLKS/J-m/Lepr db (db/db) mice were treated with combined VEGF-A165 (100 ng/wound) and PDGF-BB (50 ng/wound). Four groups were tested: fibrin only, fibrin functionalized with α 2 PI 1–8 -LAMA3 3043–3067 peptide, fibrin containing admixed GFs, and fibrin functionalized with α 2 PI 1–8 -LAMA3 3043–3067 peptide containing GFs. After 4, 7, and 10 days, a , b wound closure and c granulation tissue area were evaluated by histology (means ± SEM, day 4: n = 6, day 7: fibrin only, and α 2 PI 1–8 -LAMA3 3043–3067 peptide + GFs, n = 10; other treatment groups, n = 11, day 10: α 2 PI 1–8 -LAMA3 3043–3067 peptide, n = 8, α 2 PI 1–8 -LAMA3 3043–3067 peptide + GFs, n = 9, and other treatment groups, n = 7). b The proportions of the mice were categorized by the degree of healing after day 7 of wound treatment. d Wound histology (hematoxylin and eosin staining) at day 7. Red arrows indicate tips of the epithelium tongue. The granulation tissue (pink–violet) is characterized by a large number of granulocytes with nuclei that stain in dark-violet or black. Muscle under the wounds is stained in red. Fat tissue appears as transparent bubbles. Scale bar = 800 µm. e – g A total of 5 days after the wound treatment, e proliferation of CD31 + CD45 – endothelial cells is assessed by Ki67 + marker, and f the frequency of Ly6G + CD11b + neutrophils within CD45 + cells and g the frequency of Ly6C + CD11b + monocytes within CD45 + cells were determined using flow cytometry (means ± SEM). * P

    Journal: Nature Communications

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    doi: 10.1038/s41467-018-04525-w

    Figure Lengend Snippet: Delivering GFs and laminin HBD peptide enhances skin wound healing. Full-thickness back-skin wounds in 10– 11-week-old C57BLKS/J-m/Lepr db (db/db) mice were treated with combined VEGF-A165 (100 ng/wound) and PDGF-BB (50 ng/wound). Four groups were tested: fibrin only, fibrin functionalized with α 2 PI 1–8 -LAMA3 3043–3067 peptide, fibrin containing admixed GFs, and fibrin functionalized with α 2 PI 1–8 -LAMA3 3043–3067 peptide containing GFs. After 4, 7, and 10 days, a , b wound closure and c granulation tissue area were evaluated by histology (means ± SEM, day 4: n = 6, day 7: fibrin only, and α 2 PI 1–8 -LAMA3 3043–3067 peptide + GFs, n = 10; other treatment groups, n = 11, day 10: α 2 PI 1–8 -LAMA3 3043–3067 peptide, n = 8, α 2 PI 1–8 -LAMA3 3043–3067 peptide + GFs, n = 9, and other treatment groups, n = 7). b The proportions of the mice were categorized by the degree of healing after day 7 of wound treatment. d Wound histology (hematoxylin and eosin staining) at day 7. Red arrows indicate tips of the epithelium tongue. The granulation tissue (pink–violet) is characterized by a large number of granulocytes with nuclei that stain in dark-violet or black. Muscle under the wounds is stained in red. Fat tissue appears as transparent bubbles. Scale bar = 800 µm. e – g A total of 5 days after the wound treatment, e proliferation of CD31 + CD45 – endothelial cells is assessed by Ki67 + marker, and f the frequency of Ly6G + CD11b + neutrophils within CD45 + cells and g the frequency of Ly6C + CD11b + monocytes within CD45 + cells were determined using flow cytometry (means ± SEM). * P

    Article Snippet: Recombinant human VEGF-A165 remaining in the fibrinous matrix and in the tissue surrounding the wound was quantified by ELISA (DuoSet, R & D Systems), using 200 ng of recombinant human VEGF-A165 as 100%.

    Techniques: Mouse Assay, Staining, Marker, Flow Cytometry, Cytometry

    Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p

    Journal: Nature Communications

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    doi: 10.1038/s41467-018-04525-w

    Figure Lengend Snippet: Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p

    Article Snippet: Recombinant human VEGF-A165 remaining in the fibrinous matrix and in the tissue surrounding the wound was quantified by ELISA (DuoSet, R & D Systems), using 200 ng of recombinant human VEGF-A165 as 100%.

    Techniques: In Vitro, Cell Culture, Incubation, CyQUANT Assay

    GF retention in fibrin matrices is enhanced by incorporating laminin HBD peptide. a , b GF retention in the fibrin matrix. α 2 PI 1–8 -LAMA3 3043–3067 or α 2 PI 1–8 -LAMA5 3417–3436 peptide-functionalized fibrin matrices were made in the presence of VEGF-A165 or PDGF-BB, and incubated in eight volumes of physiological buffer for 5 days. The buffer was changed each day, and released GFs were quantified daily. The graphs show the cumulative release of a VEGF-A165 or b PDGF-BB over 5 days ( n = 4; mean ± SEM). All data points for laminin HBD peptides were statistically significant compared to controls without laminin HBD peptide ( p

    Journal: Nature Communications

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    doi: 10.1038/s41467-018-04525-w

    Figure Lengend Snippet: GF retention in fibrin matrices is enhanced by incorporating laminin HBD peptide. a , b GF retention in the fibrin matrix. α 2 PI 1–8 -LAMA3 3043–3067 or α 2 PI 1–8 -LAMA5 3417–3436 peptide-functionalized fibrin matrices were made in the presence of VEGF-A165 or PDGF-BB, and incubated in eight volumes of physiological buffer for 5 days. The buffer was changed each day, and released GFs were quantified daily. The graphs show the cumulative release of a VEGF-A165 or b PDGF-BB over 5 days ( n = 4; mean ± SEM). All data points for laminin HBD peptides were statistically significant compared to controls without laminin HBD peptide ( p

    Article Snippet: Recombinant human VEGF-A165 remaining in the fibrinous matrix and in the tissue surrounding the wound was quantified by ELISA (DuoSet, R & D Systems), using 200 ng of recombinant human VEGF-A165 as 100%.

    Techniques: Incubation

    Laminin binds promiscuously to GFs and chemokines. a Binding of multiple isoforms of full-length laminin (–111, –211, –332, –411, –421, –511, and –521) to GFs and CXCL chemokines were measured by ELISA. A450 nm represents absorbance at 450 nm. BSA-coated wells served as negative controls ( n = 4, mean ± SEM). Signals greater than 0.1 (gray box) are considered to be significant. b Affinities ( K D values are shown) of full-length laminin against VEGF-A165, PlGF-2, and PDGF-BB were measured by SPR. A SPR chip was functionalized with laminin-521 (~2000 resonance units (RU)), and each GF was flown over the chip at indicated concentrations. Curves represent the specific responses (in RU) to laminin obtained. Experimental curves were fitted with Langmuir binding kinetics. Binding kinetics values [dissociation constants ( K D ) and rate constants ( K on and K off )] determined from the fitted curves are shown. Two experimental replicates

    Journal: Nature Communications

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    doi: 10.1038/s41467-018-04525-w

    Figure Lengend Snippet: Laminin binds promiscuously to GFs and chemokines. a Binding of multiple isoforms of full-length laminin (–111, –211, –332, –411, –421, –511, and –521) to GFs and CXCL chemokines were measured by ELISA. A450 nm represents absorbance at 450 nm. BSA-coated wells served as negative controls ( n = 4, mean ± SEM). Signals greater than 0.1 (gray box) are considered to be significant. b Affinities ( K D values are shown) of full-length laminin against VEGF-A165, PlGF-2, and PDGF-BB were measured by SPR. A SPR chip was functionalized with laminin-521 (~2000 resonance units (RU)), and each GF was flown over the chip at indicated concentrations. Curves represent the specific responses (in RU) to laminin obtained. Experimental curves were fitted with Langmuir binding kinetics. Binding kinetics values [dissociation constants ( K D ) and rate constants ( K on and K off )] determined from the fitted curves are shown. Two experimental replicates

    Article Snippet: Recombinant human VEGF-A165 remaining in the fibrinous matrix and in the tissue surrounding the wound was quantified by ELISA (DuoSet, R & D Systems), using 200 ng of recombinant human VEGF-A165 as 100%.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Chromatin Immunoprecipitation

    Excess heparin inhibits GF–laminin binding. Inhibition of GF-binding to laminin (–111, –211, –221, –411, –421, –511, and –521) by excess heparin. ELISA plates were coated with 10 µg/mL laminin and further incubated with 1 μg/mL a VEGF-A165, b PlGF-2, or c FGF-2 solution in the absence or presence of excess (10 μM) heparin. Bound GFs were detected using a specific antibody for each GF ( n = 4, mean ± SEM). Statistical analyses were performed using the Mann–Whitney U test by comparing the signals with and without heparin. * p

    Journal: Nature Communications

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    doi: 10.1038/s41467-018-04525-w

    Figure Lengend Snippet: Excess heparin inhibits GF–laminin binding. Inhibition of GF-binding to laminin (–111, –211, –221, –411, –421, –511, and –521) by excess heparin. ELISA plates were coated with 10 µg/mL laminin and further incubated with 1 μg/mL a VEGF-A165, b PlGF-2, or c FGF-2 solution in the absence or presence of excess (10 μM) heparin. Bound GFs were detected using a specific antibody for each GF ( n = 4, mean ± SEM). Statistical analyses were performed using the Mann–Whitney U test by comparing the signals with and without heparin. * p

    Article Snippet: Recombinant human VEGF-A165 remaining in the fibrinous matrix and in the tissue surrounding the wound was quantified by ELISA (DuoSet, R & D Systems), using 200 ng of recombinant human VEGF-A165 as 100%.

    Techniques: Binding Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Incubation, MANN-WHITNEY

    GFs bind to laminin HBD derived from LAMA3, LAMA4, and LAMA5. a The location of laminin-derived peptides in the LG domain of LAMA3, LAMA4, and LAMA5 chains. b–f Affinity of heparin and GFs against chemically synthesized peptides derived from the LG domain of LAMA3, LAMA4, and LAMA5 chains. ELISA plates were coated with 10 µg/mL laminin peptide and further incubated with b biotinylated heparin, c VEGF-A165 and VEGF-A121, d PlGF-2 and PlGF-1, e PDGF-BB, or f FGF-2. Concentrations were 1 μg/mL for GFs and 10 µg/mL for heparin. Bound heparin was detected with streptavidin, and bound GFs with a specific antibody for each GF ( n = 4, mean ± SEM). Statistical analyses were performed using the Mann–Whitney U test by comparing the signals obtained from the laminin peptide- and the BSA-coated wells. * p

    Journal: Nature Communications

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    doi: 10.1038/s41467-018-04525-w

    Figure Lengend Snippet: GFs bind to laminin HBD derived from LAMA3, LAMA4, and LAMA5. a The location of laminin-derived peptides in the LG domain of LAMA3, LAMA4, and LAMA5 chains. b–f Affinity of heparin and GFs against chemically synthesized peptides derived from the LG domain of LAMA3, LAMA4, and LAMA5 chains. ELISA plates were coated with 10 µg/mL laminin peptide and further incubated with b biotinylated heparin, c VEGF-A165 and VEGF-A121, d PlGF-2 and PlGF-1, e PDGF-BB, or f FGF-2. Concentrations were 1 μg/mL for GFs and 10 µg/mL for heparin. Bound heparin was detected with streptavidin, and bound GFs with a specific antibody for each GF ( n = 4, mean ± SEM). Statistical analyses were performed using the Mann–Whitney U test by comparing the signals obtained from the laminin peptide- and the BSA-coated wells. * p

    Article Snippet: Recombinant human VEGF-A165 remaining in the fibrinous matrix and in the tissue surrounding the wound was quantified by ELISA (DuoSet, R & D Systems), using 200 ng of recombinant human VEGF-A165 as 100%.

    Techniques: Derivative Assay, Synthesized, Enzyme-linked Immunosorbent Assay, Incubation, MANN-WHITNEY

    GFs bind to LG domain derived from LAMA3, LAMA4, and LAMA5. Affinity of GFs against recombinant laminin LG domains. ELISA plates were coated with 1 µg/mL a LAMA3 2928–3150 , b LAMA4 826–1816 , or c LAMA5 3026–3482 and further incubated with 1 μg/mL of VEGF-A165, VEGF-A121, PlGF-2, PlGF-1, PDGF-BB, or FGF-2 solution. Bound GFs were detected using a specific antibody for each GF ( n = 4, mean ± SEM). Statistical analyses were performed using the Mann–Whitney U test by comparing the signals obtained from the laminin domain- and the BSA-coated wells. * p

    Journal: Nature Communications

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    doi: 10.1038/s41467-018-04525-w

    Figure Lengend Snippet: GFs bind to LG domain derived from LAMA3, LAMA4, and LAMA5. Affinity of GFs against recombinant laminin LG domains. ELISA plates were coated with 1 µg/mL a LAMA3 2928–3150 , b LAMA4 826–1816 , or c LAMA5 3026–3482 and further incubated with 1 μg/mL of VEGF-A165, VEGF-A121, PlGF-2, PlGF-1, PDGF-BB, or FGF-2 solution. Bound GFs were detected using a specific antibody for each GF ( n = 4, mean ± SEM). Statistical analyses were performed using the Mann–Whitney U test by comparing the signals obtained from the laminin domain- and the BSA-coated wells. * p

    Article Snippet: Recombinant human VEGF-A165 remaining in the fibrinous matrix and in the tissue surrounding the wound was quantified by ELISA (DuoSet, R & D Systems), using 200 ng of recombinant human VEGF-A165 as 100%.

    Techniques: Derivative Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, MANN-WHITNEY